The melanocyte can give rise to a number of morphologically different tumors. Most of them are biologically benign and are referred to as melanocytic nevi. Examples of melanocytic nevi are congenital nevi, Spitz nevi (including pigmented spindle cell nevi, which are regarded as a subtype of Spitz nevi), dysplastic or Clark""s nevi, blue nevi, lentigo simplex, and deep penetrating nevus.
Spitz nevi are benign melanocytic neoplasms that can have considerable histological resemblance to melanoma. They were first described as xe2x80x9cjuvenile melanomaxe2x80x9d by Sophie Spitz in 1948 and initially regarded as a subset of childhood melanoma that follows a benign course (Spitz, S., Am. J. Pathol. 24, 591-609 (1948)). Spitz nevi are common and account for about 1% of surgically removed nevi (Casso et al., J Am Acad Dermatol., 27, 901-13 (1992)). Although in general the pathological diagnosis of Spitz nevus is straightforward, there is a subset of cases in which it is difficult to impossible to histologically differentiate Spitz nevi from melanoma because of overlapping histological features, such as the presence of melanocytes with abundant cytoplasm and/or melanocytes with large pleomorphic nuclei. Additionally, mitotic figures, sometimes numerous, occur in both neoplasms.
Melanoma refers to malignant neoplasms of melanocytes. Accurate diagnosis and early treatment is of great importance because, although advanced melanoma has a poor prognosis, most melanomas are curable if excised in their early stages. Although in general the histopathological diagnosis of melanoma is straightforward, there is a subset of cases in that it is difficult to differentiate melanomas from benign neoplasm of melanocytes (LeBoit, P. E. SIMLULANTS OF MALIGNANT MELANOMA: A ROGUE""S GALLERY OF MELANOCYTIC AND NON-MELANOCYTIC IMPOSTERS, In Malignant Melanoma and Melanocytic Neoplasms, P. E. Leboit, ed. (Philadelphia: Hanley and Belfus), pp. 195-258 (1994)). Even though the diagnostic criteria for separating the many simulators of melanoma are constantly refined, a fraction of cases remains where an unambiguous diagnosis cannot be reached (Farmer et al., DISCORDANCE IN THE HISTOPATHOLOGIC DIAGNOSIS OF MELANOMA AND MELANOCYTIC NEVI BETWEEN EXPERT PATHOLOGISTS, Human Pathol. 27: 528-31 (1996)). The most frequent and important diagnostic dilemma is the differential diagnosis between Spitz nevus and melanoma.
Misdiagnosis of Spitz nevus as melanoma and vice versa has been repeatedly reported in the literature (Goldes et al., Pediatr. Dermatol., 1: 295-8 (1984); Okun, M. R. Arch. Dermatol. 115: 1416-1420 (1979); Peters et al., Histopathology, 10, 1289-1302 (1986)). A retrospective study of 102 melanomas of childhood found that only 60 cases were classified as melanoma by a panel of experts, the majority of the remainder being classified as Spitz nevi (Spatz, S., Int. J. Cancer 68, 317-24 (1996)). The presence of this diagnostic gray zone has even led the authors of a review article in the xe2x80x9cContinuing Medical Educationxe2x80x9d section of the Journal of the American Association of Dermatology to conclude that Spitz nevus and melanoma may xe2x80x9cactually exist on a continuum of diseasexe2x80x9d (Casso et al., J. Am. Acad. Dermatol., 27, 901-13 (1992)). The authors recommended that xe2x80x9ctreatment include complete excision of all Spitz nevi followed by reexcision of positive margins if present.xe2x80x9d The need for improved diagnostics for melanocytic neoplasms has led to numerous attempts to improve diagnostic accuracy by the use of markers that could be detected by immuno-histochemistry. While there have been prior efforts aimed at resolving this problem, none have been satisfactory. For example, even though tests employing markers such as S100, HMB45 are useful in establishing that a poorly differentiated tumor is of melanocytic lineage, adjunctive techniques have been of little help in separating benign from malignant melanocytic lesions.
Thus, there exists a great need for improved and accurate diagnostic methods to distinguish Spitz nevi from malignant melanoma. Furthermore, there is a need to distinguish melanocytic neoplasms that fall between Spitz nevi and malignant and are difficult to classify. The present invention addresses these and other needs by providing methods of typing a melanocytic neoplasm by detecting in a tumor sample the presence of an increase in copy number of an 11 p chromosome arm, particularly, detecting the presence of an 11p isochromosome, which indicates the presence of a Spitz nevus. Typing can also be performed by determining the presence of a mutated H-RAS gene, which is also associated with, or indicates the presence of a Spitz nevus.
The present invention provides for methods of typing a melanocytic neoplasm by detecting the presence of a mutated H-RAS gene in a patient sample, whereby the presence of the mutation in the H-RAS gene indicates the presence of a Spitz nevus, or classification as a Spitz nevus. Frequently, the mutation in the H-RAS gene is at codon 12, 13, or 61.
Typically, the copy number of the entire chromosome 11p including the H-RAS gene is increased relative to normal in the patient sample. The increase in gene copy number is frequently due to the presence of an 11p isochromosome.
In one embodiment of the invention, the presence of a mutation in the H-RAS gene is detected by amplifying a nucleic acid that encodes H-RAS or a fragment, and sequencing the amplified product to determine whether the sequence contains a mutation relative to a normal H-RAS sequence. Amplification is typically performed using PCR. Primers for the PCR reaction include those set out in SEQ ID NOs: 1 and 2, and SEQ ID NOs: 3 and 4. The nucleic acid that is amplified can be genomic DNA or RNA.
In another aspect of the invention, the presence of a mutation in the H-RAS gene is detected by contacting a nucleic acid from a skin tumor sample with a probe that selectively hybridizes to a target nucleic acid comprising an H-RAS gene to form a stable hybridization complex. The probe is contacted under condition in which the probe binds selectively to the target nucleic acid that includes the H-RAS gene. In one embodiment, the probe binds selectively to a mutated H-RAS gene. The method can further include a step of amplifying the nucleic acid from the sample. Preferably, the amplifying step is a PCR reaction, which can be performed, e.g., using oligonucleotides as set out in SEQ ID NOs: 1 and 2, and 3 and 4. The nucleic acid from the sample is preferably genomic DNA or RNA.
The invention also includes a method of detecting the presence of a mutated H-RAS gene by detecting a polypeptide encoded by the mutant H-RAS gene. Preferably the amount of polypeptide is quantified using an immunoassay, e.g., ELISA. In one embodiment, the polypeptide is detected using an antibody that selectively binds to the polypeptide encoded by the mutant H-RAS gene.
The methods of the invention further include a method of typing a patient melanocytic neoplasm from a patient by detecting the presence of an increase in copy number of the 11p chromosome arm whereby the presence of the increase in copy number of the 11p arm is indicates the presence of a Spitz nevus, or diagnosis of Spitz nevus. Typically, the methods comprise detecting the presence of an 11p isochromosome in a sample from a patient.
In one embodiment, the 11p isochromosome is detected by hybridizing a nucleic acid from the skin tumor sample with a probe that selectively hybridizes to sequences on chromosome 11p that are adjacent to the centromere and detecting the presence of one or more pairs of hybridization signals compared to normal. Additionally, the method can include hybridization of the nucleic acid sample with a second probe that is labeled with a second label distinguishable from the first and selectively hybridizes to a target nucleic acid sequence at chromosome 11q adjacent to the centromere; and detecting the presence of at least one pair of hybridization signals that consists of a signal from each of the probes. The probes are often labeled with fluorescent labels of different colors.
To facilitate understanding the invention, a number of terms are defined below.
The terms xe2x80x9cmelanomaxe2x80x9d or xe2x80x9ccutaneous melanomaxe2x80x9d refer to malignant neoplasms of melanocytes, which are pigment cells present normally in the epidermis and sometimes in the dermis. There are four types of cutaneous melanoma: lentigo maligna melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral lentiginous melanoma (AM). Melanoma usually starts as a proliferation of single melanocytes at the junction of the epidermis and the dermis. The cells first grow in a horizontal manner and settle an area of the skin that can vary from a few millimeters to several centimeters. As noted above, in most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician.
The term xe2x80x9cmelanocytic neoplasmxe2x80x9d refers to an accumulation of melanocytes that can undergo a benign, locally aggressive, or malignant course. xe2x80x9cMelanocytic neoplasmxe2x80x9d encompasses both benign melanocytic neoplasms, xe2x80x9cnevixe2x80x9d, and malignant melanocytic neoplasms, xe2x80x9cmelanomaxe2x80x9d.
The terms xe2x80x9cSpitz nevixe2x80x9d or xe2x80x9cSpitz nevusxe2x80x9d refer to melanocytic neoplasms that can have considerable histological resemblance to melanoma. They generally are benign, but can recur locally, or rarely, spread to the lymph nodes. They were first described as xe2x80x9cjuvenile melanomaxe2x80x9d and initially were thought of as a subset of childhood melanoma that follows a benign course. Spitz nevi are common and account for about 1% of surgically removed nevi.
The terms xe2x80x9ctumorxe2x80x9d or xe2x80x9ccancerxe2x80x9d in an animal refers to the presence of cells possessing characteristics such as atypical growth or morphology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal. xe2x80x9cTumorxe2x80x9d includes both benign and malignant neoplasms.
The phrase xe2x80x9ctypingxe2x80x9d or xe2x80x9cdetectingxe2x80x9d a neoplasm refers to the determination whether the neoplasm is, or has a high probability of being, a certain class of neoplasm. Classification can be based on whether the neoplasm is benign or malignant, or type of nevus, e.g., Spitz nevus. xe2x80x9cTypingxe2x80x9d or xe2x80x9cdetectingxe2x80x9d can also refer to obtaining indirect evidence regarding the likelihood of the presence of a Spitz nevus or melanoma in the patient. Detection of a Spitz nevus versus a melanoma can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the patient.
The terms xe2x80x9chybridizing specifically toxe2x80x9d, xe2x80x9cspecific hybridizationxe2x80x9d, and xe2x80x9cselectively hybridize to,xe2x80x9d as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. A xe2x80x9cstringent hybridizationxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I, Ch. 2, xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assays,xe2x80x9d Elsevier, N.Y. (xe2x80x9cTijssenxe2x80x9d). Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42xc2x0 C. using standard hybridization solutions (see, e.g., Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, e.g., Sambrook supra.) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4xc3x97 to 6xc3x97SSC at 40xc2x0 C. for 15 minutes.
The term xe2x80x9clabeled with a detectable labelxe2x80x9d, as used herein, refers to a nucleic acid attached to a detectable composition, i.e., a label. The detection can be by, e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or chemical means. For example, useful labels include 32P, 35S, 3H, 14C, 125I, 131I; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense reagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. Dynabeads(trademark)), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label can be directly incorporated into the nucleic acid, peptide or other target compound to be detected, or it can be attached to a probe or antibody that hybridizes or binds to the target. Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield, Mol Cell Probes 9: 145-156 (1995). In addition, target DNA sequences can be detected by means of the primed in situ labeling technique (PRINS) (Koch et al., Genet. Anal. Tech. Appl. 8: 171-8, (1991)). The sensitivity of the detection can be increased by using chemical amplification procedures, e.g., by using tyramide (Speel et al., J. Histochem. Cytochem. 45:1439-46, (1997)).
The term xe2x80x9cpaired hybridization signalsxe2x80x9d or a xe2x80x9chybridization signal pairxe2x80x9d refers to a spatial pattern of hybridization signals wherein two signals are consistently identified in close proximity. Isochromosomes are typically characterized by the presence of xe2x80x9cpaired hybridization signalsxe2x80x9d from a single probe. For example, in a sample with many cells, a xe2x80x9chybridization signal pairxe2x80x9d is a consistent occurrence of two signals in close proximity that is clearly not due to an artifact or a random event.
The term xe2x80x9cnucleic acidxe2x80x9d as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3xe2x80x2-thioacetal, methylene(methylimino), 3xe2x80x2-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Sainstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
The term a xe2x80x9cnucleic acid arrayxe2x80x9d as used herein is a plurality of target elements, each target element comprising one or more nucleic acid molecules (probes) immobilized on one or more solid surfaces to which sample nucleic acids can be hybridized. The nucleic acids of a target element can contain sequence(s) from specific genes or clones, e.g. from specific regions of chromosome 11p or 11q. Other target elements will contain, for instance, reference sequences. Target elements of various dimensions can be used in the arrays of the invention. Generally, smaller, target elements are preferred. Typically, a target element will be less than about 1 cm in diameter. Generally element sizes are from 1 xcexcm to about 3 mm, preferably between about 5 xcexcm and about 1 mm. The target elements of the arrays may be arranged on the solid surface at different densities. The target element densities will depend upon a number of factors, such as the nature of the label, the solid support, and the like. One of skill will recognize that each target element may comprise a mixture of nucleic acids of different lengths and sequences. Thus, for example, a target element may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths. The length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure, and to provide the required resolution among different genes or genomic locations.
The terms xe2x80x9cnucleic acid samplexe2x80x9d or xe2x80x9csample of human nucleic acidxe2x80x9d as used herein refers to a sample comprising human DNA or RNA in a form suitable for detection by hybridization or amplification. Typically, it will be prepared from a skin tissue sample of a tumor from a patient who has or is suspected of having a melanocytic tumor that may be difficult to classify.
The nucleic acid sample can often be a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared using standard techniques such that individual chromosomes remain substantially intact. Alternatively, the nucleic acid may be isolated, cloned or amplified. It can be, e.g., genomic DNA, mRNA, or cDNA or selected H-RAS sequences (e.g. the promoter, particular exons, or subsequences of the gene, etc.).
The nucleic acid sample is typically extracted from particular cells, e.g. melanocytes, or prepared from a skin tumor, i.e., a melanocytic neoplasm. Methods of isolating cell and tissue samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, needle biopsies, and the like. Frequently the sample will be a xe2x80x9cclinical samplexe2x80x9d which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological purposes. The sample can also be derived from extracts or supernatants from the cells or the cells themselves from cell cultures, cells from tissue culture and other media in which it may be desirable to detect chromosomal abnormalities, such as changes in copy number, isochromosomes, or H-RAS gene mutations. In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample may be isolated nucleic acids immobilized on a solid.
The term xe2x80x9cprobexe2x80x9d or xe2x80x9cnucleic acid probexe2x80x9d, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected. The probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the regions described herein. The probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. The word xe2x80x9csamplexe2x80x9d may be used herein to refer not only to detected nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, e.g., with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately.
A probe that is xe2x80x9cadjacent to the centromerexe2x80x9d refers to a probe that hybridize to regions adjacent to the centromere bind to sequences at 11p11.1 to 11p11.2 or 11q11.1 to 11q11.2.
An xe2x80x9c11p chromosome armxe2x80x9d is defined cytogeneticallya s encompassing the chromosome from band 11p11 to 11pter.
What xe2x80x9cprobexe2x80x9d refers to specifically is clear from the context in which the word is used. The probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. In some embodiments, the probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854). One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are xe2x80x9csubstantially identicalxe2x80x9d to the disclosed probes, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived (see discussion above). Such modifications are specifically covered by reference to the individual probes described herein.
The term xe2x80x9cimmunoassayxe2x80x9d is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
The phrase xe2x80x9cspecifically (or selectively) bindsxe2x80x9d to an antibody or xe2x80x9cspecifically (or selectively) immunoreactive with,xe2x80x9d when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background, more typically more than 10 to 100 times background, and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular H-RAS protein. For example, an antibody that selectively binds to a polypeptide encoded by a mutated H-RAS gene binds to mutated, but not normal H-RAS.
xe2x80x9cProviding a nucleic acid samplexe2x80x9d means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g. isolated by another person).
xe2x80x9cTissue biopsyxe2x80x9d refers to the removal of a biological sample for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.